Fig. 1.

USP28 regulates MAST1 protein levels. A Transfection of an entire set of sgRNAs targeting individual USP subfamily genes along with Cas9 nuclease into cisplatin-resistant HeLa cells (HeLa-cisR). Transfected cells were treated with a sub-lethal dose (5 µg/mL) of cisplatin. HeLa-cisR cells treated with saline served as the negative control (vehicle) and cisplatin-treated HeLa-cisR cells co-transfected with scrambled sgRNA and Cas9 served as the mock control. Cisplatin-induced cell death was estimated using a cell viability assay and represented as a graph. Data are presented as the mean and standard deviation of three independent experiments (n = 3). B The USP28-depleted cells were treated with an increasing concentrations of cisplatin (5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL and 25 µg/mL) for 48 h, and cell viability was measured. The IC₅₀ values of cisplatin in HeLa-CisR mock and USP28-sgRNA transfected cells were 6.32 µg/mL and 3.97 µg/mL, respectively. C Schematic representation of the sgRNAs targeting exon 2 of USP28 gene. The red arrowheads indicate the positions of sgRNAs target site on the sense DNA strand. PAM sequences are indicated in bold blue font; USP28 sgRNA sequences are indicated in red font. D The validation of efficiency of sgRNAs targeting USP28 by transient co-transfection with Cas9 in HEK293 cells and immunoblotting with USP28 antibody. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control (USP28/GAPDH) and presented below the blot. The effect of depleting USP28 on endogenous MAST1 protein was estimated in HEK293 cells. E Validation of sgRNA efficiency targeting USP28 gene by transient co-transfection with Cas9 and sgRNA1 or sgRNA2 into HEK293 cells followed by a T7E1 assay to determine the cleavage efficiency. The cleaved band intensity (indel %) obtained by T7E1 assay was measured using ImageJ software and indicated. Scrambled sgRNA transfected HEK293 cells were used as a control cells. The black arrowhead indicates the cleaved PCR amplicons. A549 cells were transfected with increasing concentrations of F Flag-USP28 and G Flag-USP28CA to validate its effect on endogenous MAST1 protein levels. H The effect of reconstitution of Flag-USP28 on endogenous MAST1 protein in USP28-depleted A549 cells was validated. The protein band intensities for F–H were estimated using ImageJ software with reference to the GAPDH control band (MAST1/GAPDH) and presented below the blot. HEK293 cells were transfected with constant amount of Myc-MAST1 and increasing concentrations of I Flag-USP28 and J Flag-USP28CA to validate its effect on exogenous Myc-MAST1 protein levels. K The effect of reconstitution of Flag-USP28 on Myc-MAST1 protein in USP28-depleted HEK293 cells was validated. The protein band intensities for I–K were estimated using ImageJ software with reference to the GAPDH control band (Myc-MAST1/GAPDH) and presented below the blot